Methods for the treatment of an infectious bacterial disease with an anti-lactone or lactone derived signal molecules antibody

ABSTRACT

The present invention relates to methods for the control of virulence of infectious bacteria by modulating the extra-cellular concentration of bacterial cell signalling molecules. Derivatives of cell signalling molecules are conjugated to suitable carrier proteins and used to isolate high affinity receptors recognizing the native signal molecule(s). By binding to signalling molecules, the receptors reduce and maintain extra-cellular concentrations of signal molecules below the threshold level that would otherwise result in certain opportunistic pathogens adopting a virulent form, and can transform virulent organisms to non-virulent states. These receptors have applications for the treatment of individuals with susceptibility to infection, the treatment of patients with existing infections, in disease monitoring and management, and in related applications where the host for infection is an animal or plant.

FIELDS OF THE INVENTION

The present invention relates to methods for controlling and treatingbacterial infections in patients. The methods of the invention areapplicable to most, if not all gram negative and grain-positivebacterial infections. The invention provides for the application oftherapies based upon, in the preferred embodiment, immunoglobulin orimmunoglobulin-like receptor -molecules that have affinity andspecificity for signalling molecules involved in the processes ofbacterial cell to cell communication. By binding to such molecules, thereceptors can be used to diagnose the presence of bacteria or to assessthe disease state of patients, and can further be used to controlconcentrations of molecules involved in inducing a virulent state inopportunistic and other pathogens.

BACKGROUND OF THE INVENTION

One of the major causes of mortality and morbidity amongst patientsundergoing treatment in hospitals today is due to hospital acquiredinfection. Susceptibility to such infection can be as a result of theprimary illness for which the patient was admitted, ofimmuno-suppressive treatment regimes, or as a consequence of injuryresulting in serious skin damage, such as bums. The bacterium to whichthe highest proportion of cases is attributed is Pseudomonas aeruginosa.It is the epitome of an opportunistic pathogen of humans. The bacteriumalmost never infects uncompromised tissues, yet there is hardly anytissue that it cannot infect, if the tissue defences are compromised insome manner. Although accounting for a relatively small number ofspecies, it poses a serious threat to human health and is used hereafteras a representative example of an infectious bacterium, and does not inany way limit the scope or extent of the present invention.

Ps. aeruginosa is an opportunistic pathogen that causes urinary tractinfections, respiratory system infections, dermatitis, soft tissueinfections, bacteraemia and a variety of systemic infections,particularly in victims of severe bums, and in cancer and AIDS patientswho are immunosuppressed. Respiratory infections caused by Ps.aeruginosa occur almost exclusively in individuals with a compromisedlower respiratory tract or a compromised systemic defence mechanism.Primary pneumonia occurs in patients with chronic lung disease andcongestive heart failure. Bacteraemic pneumonia commonly occurs inneutropenic cancer patients undergoing chemotherapy. Lower respiratorytract colonisation of cystic fibrosis patients by mucoid strains of Ps.aeruginosa is common and difficult, if not impossible, to treat.

It causes bacteraemia primarily in immuno-compromised patients.Predisposing conditions include haematologic malignancies,immuno-deficiency relating to AIDS, neutropenia, diabetes mellitus, andsevere burns. Most Pseudomonas bacteraemia is acquired in hospitals andnursing homes where it accounts for about 25 percent of all hospitalacquired gram-negative bacteraemias.

The bacterium is notorious for its natural resistant to many antibioticsdue to the permeability barrier afforded by its outer membrane LPS andis, therefore, a particularly dangerous and dreaded pathogen. Also, itstendency to colonise surfaces in a biofilm form makes the cellsimpervious to therapeutic concentrations of antibiotics. Since itsnatural habitat is the soil, living in association with the bacilli,actinomycetes and moulds, it has developed resistance to a variety oftheir naturally occurring antibiotics. Moreover, Pseudomonas spp.maintain antibiotic resistance plasmids, both Resistance factors(R-factors) and Resistance Transfer Factors (RTFs), and arc able totransfer these genes by means of the bacterial processes of transductionand conjugation. Only a few antibiotics are effective againstPseudomonas, including fluoroquinolone, gentamicin and imipenem, andeven these antibiotics are not effective against all strains.Combinations of gentamicin and carbenicillin are reportedly effective inpatients with acute Ps. aeruginosa infections. The futility of treatingPseudomonas infections with antibiotics is most dramatically illustratedin cystic fibrosis patients, virtually all of whom eventually becomeinfected with a strain that is so resistant it cannot be treated.Because of antibiotic resistance, susceptibility testing of clinicalisolates is mandatory.

Ps. aeruginosa can usually be isolated from soil and water, as well asthe surfaces of plants and animals. It is found throughout the world,wherever these habitats occur, so it is quite a “cosmopolitan”bacterium. It is sometimes present as part of the normal flora ofhumans, although the prevalence of colonisation of healthy individualsoutside the hospital is relatively low (estimates range from 0 to 24percent depending on the anatomical locale). In hospitals it is known tocolonise food, sinks, taps, mops, respiratory equipment surgicalinstruments. Although colonisation usually precedes infections by Ps.aeruginosa, the exact source and mode of transmission of the pathogenare often unclear because of its ubiquitous presence in the environment.Amongst intensive care patients in whom infection is suspected onclinical grounds, as many as 50% have no identifiable source forinfection. Currently 1,400 deaths worldwide are caused each day by Ps.aeruginosa in intensive care units (ICU's), making it the No 1 killer.

Ps. aeruginosa is primarily a nosocomial pathogen. According to the CDC,the overall incidence of Ps. aeruginosa infections in US hospitalsaverages about 0.4 percent (4 per 1000 discharges), and the bacterium isthe fourth most commonly isolated nosocomial pathogen accounting for10.1% of all hospital-acquired infections. Globally it is responsiblefor 16% of nosocomial pneumonia cases, 12% of acquired urinary tractinfections, 8% of surgical wound infections and 10% of bloodstreaminfections. Immuno-compromised patients such as neutropenic cancer andbone marrow transplant patients are susceptible to opportunistic Ps.aeruginosa infection, leading to 30% reported deaths. It is alsoresponsible for 38% of ventilator-associated pneumonias and 50% ofdeaths amongst AIDS patients. In burns cases Ps. aeruginosa infectionshave declined in recent years due to improved treatment and dietarychanges. Mortality rates however remain high, accounting for 60% alldeaths due to secondary infection of burns patients.

One reason for the versatility of Ps. aeruginosa is that it produces adiverse battery of virulence determinants including elastase, LasAprotease, alkaline protease, rhamnolipids, type IV pilus-mediatedtwitching motility, pyoverdin (Williams et al., 1996, Stintzi et al.,1998, Glessner et al., 1999), pyocyanin (Brint & Ohman, 1995, Reimmannet al., 1997) and the cytotoxic lectins PA-I and PA-II (Winzer et al.,2000). It is now known that many of these virulence determinants areregulated at the genetic level in a cell density-dependent mannerthrough quorum sensing. Ps. aeruginosa possesses at least two quorumsensing systems, namely the las and rhl (vsm) systems which comprise ofthe LuxRI homologues LasRI (Gambello & Iglewski, 1991) and RhlRI (VsmRI)(Latifi et al., 1995) respectively (FIG. 2). LasI directs the synthesisof 3-oxo-C12-HSL (Passador et al., 1993, Pearson et al., 1994) whereasRhlI directs the synthesis of C4-HSL (Winson et al., 1995). The las andthe rhl systems are thought to exist in a hierarchy where the las systemexerts transcriptional control over RhlR (Williams et al., 1996, Pesciet al., 1997). The transcriptional activator LasR functions inconjunction with 3-oxo-C12-HSL to regulate the expression of the genesencoding for the virulence determinants elastase, LasA protease,alkaline protease and exotoxin A (Gambello & Iglewski, 1991, Toder etal., 1991, Gambello et al., 1993, Pearson et al., 1994) as well as lasI.Elastase is able to cleave collagen, IgG and IgA antibodies, complement,and facilitates bacterial adhesion onto lung mucosa. In combination withalkaline protease it also causes inactivation of gamma Interferon (INF)and Tumour Necrosis Factor (TNF). LasI directs the synthesis of3-oxo-C12-HSL which together with LasR, binds to the lasI promoter andcreates a positive feedback system. The RhIR transcriptional activator,along with its cognate AHL (C4-HSL), regulates the expression of rhlAB(rhamnolipid), lasB, aprA, RpoS, cyanide, pyocyanin and the lectins PA-Iand PA-II (Ochsner et al., 1994, Brint & Ohman, 1995, Latifi et al.,1995, Pearson et al., 1995, Winson et al., 1995, Latifi et al., 1996,Winzer et al., 2000). These exist in a hierarchical manner where by theLasR/3-oxo-C12-HSL regulates rhlR (Latifi et al., 1996, Pesci et al.,1997) and consequently both systems are required for the regulation ofall the above virulence determinants.

A number of different approaches are being actively pursued to developtherapeutics for the treatment or prevention of Ps. aeruginosainfection. Some are intended to be broad ranging while others aredirected at specific types of Pseudomonas infection. Those that followtraditional routes include the development of vaccines such as thatdescribed in U.S. Pat. No. 6,309,651, and a new antibiotic drug (SLIT)that is hoped will be effective against gram-negative bacteria ingeneral but is designed primarily to act against Ps. aeruginosa and isadministered by aerosol inhalation. A further observation underinvestigation is that the antibiotic erythromycin administered atsub-optimal growth inhibitory concentrations simultaneously suppressesthe production of Ps. aeruginosa haemagglutinins, haemolysin, proteasesand homoserine lactones (HSLs), and may be applicable for the treatmentof persistent Ps. aeruginosa infection. Cream formulations containingamphipathic peptides are also being examined as a possible means ofpreventing infection of bums or other serious skin wounds. U.S. Pat. No.6,309,651 also teaches that antibodies against the PcrV virulenceprotein of Ps. aeruginosa may afford protection against infection.

There is also some interest in the modulation of homoserine lactonelevels as a means of controlling pathogenicity. Certain algae have beendemonstrated to produce competitive inhibitors of acyl-homoserinelactones (AHL's) such as furanones (Masefield, 1999), as have someterrestrial plants. These compounds displace the AHL signal moleculefrom its receptor protein and can act as agonist or antagonist in AHLbioassays (Tepletski et al., 2000). Other methods employed to reduce HSLconcentration include the development of auto-inducer inactivationenzymes (AiiA's) that catalyse the degradation of HSLs.

There are a number of potential problems and limitations associated withthe therapies currently under development. It is as yet unproven as towhether vaccines will be efficacious treatments. Ps. aeruginosa producesan extensive mucoid capsule that effectively protects againstopsonisation by host antibodies, as revealed by patients with persistentinfections having high serum titres of anti-Pseudomonas antibodies. Alimitation in the applicability of treatments such as vaccines andanti-PcrV antibodies, as described in U.S. Pat. No. 6,309,651, is thatthese approaches restrict themselves to Pseudomonas infection, and wouldnot be efficacious against other bacteria. The use of auto-inducermimics are limited by the concentrations of most that are required toeffectively compete against HSLs for the receptor binding site, and thepossibility of side effects. It is well known that HSLs released byPseudomonas and other bacteria have a number of direct effects on humanphysiology. These include inhibition of histamine release as describedin WO 01/26650. WO 01/74801 describes that HSLs are also able to inhibitlymphocyte proliferation and down-regulate the secretion of TNF-α bymonocytes and macrophages, so acting as a general immuno-suppressant.There is a danger therefore that therapies involving the use ofcompetitive HSL mimics may result in down-regulation of the patient'simmune system. This would be generally undesirable, and particularly soin immuno-compromised patients. The use of antibiotics can, at best, beviewed as a short-term strategy in view of the remarkable ability ofthis bacterium (and others) to develop resistance to antibiotics.

That the pathogenesis of Ps. aeruginosa is clearly multifactoral isunderlined by the large number of virulence factors and the broadspectrum of diseases associated with this bacterium. Many of theextra-cellular virulence factors required for tissue invasion anddissemination are controlled by cell-to-cell signalling systemsinvolving homoserine lactone-based signal molecules and specifictranscriptional activator proteins. These regulatory systems allow Ps.aeruginosa to adapt to a virulent form in a co-ordinated cell densitydependent manner, and to overcome host defence mechanisms. Interferencewith such cell signalling and the associated production of virulencefactors is a promising therapeutic approach to reducing illness anddeath caused by Ps. aeruginosa. The importance of such approaches ishighlighted by the growing number of bacterial pathogens found toutilise similar cell-to-cell signalling systems.

In order to study the molecular basis of host-pathogen interactions, itis desirable to have available a suitable model system (non-human) inwhich the stimuli and mechanisms relating to pathogenicity in humans canbe replicated. In the case of many diseases the pathogen concerned isintrinsically associated with one, or a few closely related species orgroups, e.g. HIV. Other organisms can cause disease in a wide range ofhost, crossing the species, genus, and even kingdom barriers. Ps.aeruginosa is one such pathogen, being able to infect a variety of bothplant, insect and animal species.

In recent years it has been demonstrated that Ps. aeruginosa strainsthat are able to cause disease in humans and mice are also able to killthe nematode worm Caemohabtidis elegans (Tan et al., 1999a, Tan et al.,1999b, Tan et al., 2000). More importantly, the pathogenicity of Ps.aeruginosa to C. elegans is regulated by the same cell density-dependantquorum sensing systems that control pathogenesis in humans. The recentcompletion of the sequencing of the genomes of both Ps. aeruginosa andC. elegans make this relationship ideal for the study of bacterialdisease mechanisms. The fact that 36% of C. elegans proteins also havehomologues in humans (Darby et al., 1999), and the ease with which C.elegans can be grown in the laboratory, have lead to its widespread useas a model for pathogenisis and host defences in humans (Kurz andEwbank, 2000).

A variety of different mechanisms by which Ps. aeruginosa mediateskilling of C. elegans have been identified. Tan et al., 1999a; 1999b,and Mahajan-Miklos et al., 1999, describe the use of a clinical isolate(strain PA14) that also infects mice and plants. By varying the growthconditions of the bacteria, subsequent application to C. elegans canresult in either fast killing (within hours) or slow killing (within 3to 4 days. The fast killing mechanism is dependant only on the Rhlquorum sensing system. Moreover, the use of cell-free medium in whichPs. aeruginosa have been appropriately grown, or heat killed extractsare equally effective as death is effected by diffusible pyocyanintoxin. In contrast the slow killing mechanism is reliant on both Las andRhl systems and results in significant infection of the nematode gut. Asdeath is probably a result of infiltration of the host by the bacteria,this assay provides the most useful nematode model for infection inanimals. A third killing mechanism has been described by Darby et al.,(1999). Here the use of Ps. aeruginosa strain PA01 (a known humanpathogen) grown in brain-heart infusion medium results in rapidparalysis and death of C. elegans. As with the slow killing describedearlier, paralysis is both Las and Rhl system-dependant.

There is a need to develop effective means of modulating theconcentrations of HSLs and other bacterial cell signalling moleculesinvolved in pathogenicity by methods that do not have adverse sideeffects, and are unlikely to be evaded by pathogenic bacteria in theforeseeable future.

SUMMARY OF THE INVENTION

The present invention provides for methods for controlling the virulenceof human, animal and plant pathogenic bacteria by regulating theextra-cellular concentrations of bacterial cell signalling molecules.Whereas other treatments are restricted to a particular pathogen orgroup of pathogens, or to specific aspects of bacterial virulence, thepresent invention addresses bacterial virulence in general.

According to a first aspect of the present invention, there is providedan antibody to a lactone or lactone-derived signal molecule secreted bybacteria.

Antibodies according to the present invention can be polyclonalantibodies or monoclonal antibodies. Polyclonal antibodies can be raisedby stimulating their production in a suitable animal host (e.g. a mouse,rat, guinea pig, rabbit, sheep, chicken, goat or monkey) when theantigen is injected into the animal. If necessary an adjuvant may beadministered together with the antigen. The antibodies can then bepurified by virtue of their binding to antigen or as described furtherbelow. Monoclonal antibodies can be produced from hybridomas. These canbe formed by fusing myeloma cells and B-lymphocyte cells which producethe desired antibody in order to form an immortal cell line. This is thewell known Kohler & Milstein technique (Nature 256 52-55 (1975)).

Techniques for producing monoclonal and polyclonal antibodies which bindto a particular protein are now well developed in the art. They arediscussed in standard immunology textbooks, for example in Roitt et al,Immunology second edition (1989), Churchill Livingstone, London.

In addition to whole antibodies, the present invention includesderivatives thereof which are capable of binding to antigen. Thus thepresent invention includes antibody fragments and synthetic constructs.Examples of antibody fragments and synthetic constructs are given byDougall et al in Tibtech 12 372-379 (September 1994). Antibody fragmentsinclude, for example, Fab, F(ab′)₂ and Fv fragments (see Roitt et al[supra]). Fv fragments can be modified to produce a synthetic constructknown as a single chain Fv (scFv) molecule. This includes a peptidelinker covalently joining V_(H) and V_(L) regions which contribute tothe stability of the molecule. The present invention therefore alsoextends to single chain antibodies or scAbs.

Other synthetic constructs include CDR peptides. These are syntheticpeptides comprising antigen binding determinants. Peptide mimetics mayalso be used. These molecules are usually conformationally restrictedorganic rings which mimic the structure of a CDR loop and which includeantigen-interactive side chains. Synthetic constructs also includechimaeric molecules. Thus, for example, humanised (or primatised)antibodies or derivatives thereof are within the scope of the presentinvention. An example of a humanised antibody is an antibody havinghuman framework regions, but rodent hypervariable regions. Syntheticconstructs also include molecules comprising a covalently linked moietywhich provides the molecule with some desirable property in addition toantigen binding. For example the moiety may be a label (e.g. adetectable label, such as a fluorescent or radioactive label) or apharmaceutically active agent.

In order to generate anti-bacterial signal molecule antibodies, it ispreferable to conjugate the target molecule, or a suitable derivative,to two different carrier molecules (proteins), though a singleconjugated species can be also used. Bacterial signal molecules, ingeneral, are too small to stimulate an immune response in-vivo, or to beused directly as a source of antigen for the selection of high affinityantibodies from antibody libraries. Selection of antibodies specific forthe cell signalling molecule (hereafter referred to as ‘antigen’) iscarried out in the preferred embodiment using a repertoire (library) offirst members of specific binding pairs (sbp), for example a library ofantibodies displayed on the surface of filamentous bacteriophage. Anyother system that allows for the selection of specific receptors from alibrary of receptors is also applicable for the methods of the presentinvention. In alternative embodiments signal molecule-specific clonescan be selected from a panel of antibody secreting hybridoma cell linesgenerated from an animal immunised with an antigen conjugate. For thepurposes of a general illustration the example of a library of antibodybinding sites displayed on phage particles will be used.

A conjugate comprising an antigen coupled to a suitable carriermolecule, which can be a protein, a peptide or any natural or syntheticcompound or material (referred to hereafter as ‘conjugate-1’) isimmobilised onto a suitable solid support such as an ‘immunotube’ ormicroliter plate, and the uncoated surface blocked with a non-specificblocking agent such as dried milk powder. Suitable conjugate moleculescan include, but are not limited to proteins such as bovine serumalbumin (BSA), Keyhole Limpet Haemocyanin (KLH), Bovine Thyroglobulin(TG), Ovalbumin (Ova), or non-proteins such as biotin. The onlyrestriction on the selection of the conjugate molecule is that it beimmobilisable in some way and for immunisation is large enough to elicitan immune response.

A library of first members of specific binding pairs (sbp's) (‘thelibrary’) is applied to the immobilised conjugate and incubated forsufficient time for sbp members recognising conjugate-1 to bind. Phagenot recognising the conjugate are removed by stringent washing. Phagethat remain bound are eluted, for example with tri-ethylamine or othersuitable reagent, into a buffer solution to restore neutral pH.Recovered phage particles are then infected into a suitable hostorganism, e.g. E. coli bacteria, and cultured to amplify numbers of eachselected member and so generate a second ‘enriched’ library. The processis then repeated using the enriched library to select forphage-antibodies (‘phage’) recognising the antigen conjugated to asecond carrier protein (conjugate-2),

Additional rounds are performed as required, the selection process beingaltered to favour selection of those sbp members recognising the freeform of the antigen. Phage are selected against antigen conjugates asdescribed previously, using initially conjugate-1, and alternating withconjugate-2 (where available) for each subsequent round. Bound phage areelated by incubating with a solution of free antigen, or antigenconjugated to small soluble selectable moieties, e.g. biotin, forsufficient time for sbp members with higher affinity for the bound formof the antigen to dissociate from the immobilised conjugate. Those phageeluted with free antigen are infected into E. coli cells foramplification and re-selection, and those remaining bound to theimmobilised antigen discarded. Alternatively, but less preferably, allantibodies binding to conjugate may be eluted eg. with low pH.

Individual (monoclonal) phage clones from each round of selection arescreened for desired binding characteristics. This can be performed by avariety of methods that will be familiar to those with ordinary skill inthe art, depending on requirements, including such techniques as SPR(Surface Plasmon Resonance) and ELISA (Enzyme Linked Immuno-SorbantAssay). Selection criteria will include the ability to bindpreferentially to the free soluble form of the antigen in the presenceof conjugated derivatives.

In the preferred embodiment of the invention, antibodies will begenerated from a naïve human antibody phage display library (McCaffertyet al., Nature 348: 552-554, 1990; and as described in WO 92/01047).Thus the antibodies could be used for administering to patients inaddition to use as diagnostic or dialysis reagents. In a diagnosticassay the antibody could be used to determine the presence andconcentration of HSLs in patients and so predict the patient's infectionstatus. In other embodiments a library can be constructed from an animalpre-immunised with one or more conjugates of a HSL and a suitablecarrier molecule. A further alternative is the generation of hybridomacell lines from an animal immunised as described above. In the lattertwo cases it is preferable that steps be taken to reduce theimmunogenicity of resulting antibodies, for example by creating hostanimal-human chimaeric antibodies, or “humanisation” by CDR graftingonto a suitable antibody framework scaffold. Other methods applicablewill include the identification of potential T-cell epitopes within theantibody, and the subsequent removal of these e.g. by site-directedmutagenesis (de-immunisation). In a further embodiment the antibody canbe engineered to include constant regions from different classes ofhuman immunoglobulin (lgG, IgA, etc.) and produced as a whole antibodymolecule in animal cells. In particular these approaches are desirablewhere the antibodies are to be used therapeutically

For the present invention, the antibody may be monoclonal or polyclonal.The antibodies may be human or humanised, or for dialysis/diagnosticapplications may be from other species. Antibody fragments orderivatives, such as Fab, F(ab').sup.2 (also written as F(ab′)₂), Fv, orscFv, may be used, as may single-chain antibodies (scAb) such asdescribed by Huston et al. (Int. Rev. Immunol. 10: 195-217, 1993),domain antibodies (dAbs), for example a single domain antibody, orantibody-like single domain antigen-binding receptors. In addition toantibodies, antibody fragments and immunoglobulin-like molecules,peptidomimetics or non-peptide mimetics can be designed to mimic thebinding activity of antibodies in preventing or modulating bacterialinfection by inhibiting the binding of cell-signalling molecules.

After the preparation of a suitable antibody, it may be isolated orpurified by one of several techniques commonly available (for example,as described in Antibodies: A Laboratory Manual, Harlow and Lane, eds.Cold Spring Harbor Laboratory Press (1988)). Generally suitabletechniques include peptide or protein affinity columns, HPLC or RP-HPLC,purification on Protein A or Protein G columns, or combinations of thesetechniques. Recombinant antibodies can be prepared according to standardmethods, and assayed for specificity using procedures generallyavailable, including ELISA, ABC, dot-blot assays etc.

The lactone signal molecule may be a homoserine molecule, or a peptidethiolactone molecule.

The homoserine lactone molecule can have a general formula selected fromthe group consisting of:

where n=0 to 12.

Compounds of general formula I can be described as acyl-homoserinelactone molecules. Compounds of general formula II can be described as3-oxo-homoserine lactones. Compounds of general formula III can bedescribed as 3-hydroxy-homoserine lactones.

Preferred homoserine lactone molecules for general formula I areN-butanoly-L-homoserine lactone (BBL) where n=0,N-dodecanoyl-L-homoserine lactone (dDHL) where n=8 andn-tetradecanoyl-L-homoserine lactone (tDHL) where n=10. Preferredhomoserine lactone molecules for general formula II areN-(-3-oxohexanoyl)-L-homoserine lactone (OHHL) where n=2 andN-(-3-oxododecanoyl)-L-homoserine lactone (OdDHL) where n=8. Preferredhomoserine lactone molecules for general formula III areN-(-3-hydroxybutanoyl)-L-homoserine lactone (HBHL) where n=0.

In general the bacterial HSLs can be further subdivided into twoclasses: i) long chain molecules (10-12 carbons) and ii) short chainmolecules (4-8 carbons). In Pseudomonas sp these different size classesbind to different R molecules and cause different genes to be switchedon. Long chain molecules bind to the R homologue gene product known asLAS and short chain molecules to the RHL protein homologue.

The peptide thiolactone can have a general formula (IV) as follows:

where X is any amino acid and n=1 to 10.

In the above, and throughout this specification, the amino acid residuesare designated by the usual IUPAC single letter nomenclature. The singleletter designations may be correlated with the classical three letterdesignations of amino acid residues as follows:

-   -   A=Ala G=Gly M=Met S=Ser    -   C=Cys H=His N=Asn T=Thr    -   D=Asp I=Ile P=Pro V=Val    -   E=Glu K=Lys Q=Gln W=Trp    -   F=Phe L=Leu R=Arg Y=Tyr

Preferred peptide thiolactone molecules may have the followingstructures:

A growing number of bacterial species are being found to communicatebetween cells using a variety of small signal molecules. Gram-negativebacteria predominantly use N-acyl homoserine lactones (Table 1). Thelatter are a group of compounds that share a common homoserine lactonering structure and vary in the length and structure of a side chain(FIG. 1 a). There are three classes within the group, theacyl-homoserine lactones, the 3-oxo-homoserine lactones and the3-hydroxy-homoserine lactones. A single species can produce and respondto members of more than one class.

The lactone-derived signal molecule may be a furanosyl borate diester,for example, AutoInducer-2 or AI-2, Pro-AI-2 or a Pro-AI-2-reactivehapten (FIG. 1 b). Many gram negative and gram positive organisms suchas Vibrio harveyi and Bacillus anthracis produce a second signalmolecule, AI-2, that is derived from the same S-Adenosylmethioninesource as homoserine lactones, and binds to the receptor LuxP (FIG. 1b). It is thought likely that AI-2 is a universal bacterial signalmolecule, being produced and recognised by and induces virulence in awide variety of species.

AI-2 can be described as 2,3-dihydroxy-4-methyl-3,4-borate diester.

A lactone-derived signal molecule can also be a derivative of4,5-dihydroxy-2,3-pentanedione (DPD) which cyclizes naturally to formPro-AI-2, which reacts naturally with boric acid to form AI-2 (FIG. 1b). Pro-AI-2 can be described as 2,4-dihydroxy-4-methyl-furan-3-one.Pro-AI-2 can be derivatised as shown in FIG. 1 b at the 4-methylposition to add a heptanoic acid moeity to form a Pro-AI-2 reactivehapten. Other derivatives may also include other straight chain orbranched, saturated or unsaturated C₁-C₁₀ carboxylic acid moieties, suchas methanoic, ethanoic, propanoic, pentanoic, hexanoic, heptanoic,octanoic, nonanoic or decanoic acid.

Gram-positive bacteria such as Staphylococcus aureus use short peptides(FIG. 1 c) (Mayville et al., 1999). The cells use the molecules as ameans of determining the local cell density, such that in conditions oflow cell density the concentration of signal molecule is correspondinglylow. In high cell densities the local signal molecule concentration ishigh. When this concentration reaches a threshold level it induces thetranscription of genes involved in virulence and the onset of a diseasestate in the host.

The thiolactone derivatised peptide signal molecules used byStaphylococcus spp. have additional biological functions. They not onlyprovide the bacteria with information about their local populationdensity, but they also serve to suppress virulence in other S. aureusbelonging to different sub-groups (Lyon et. al., 2000). Thisbi-functionality is split between the different structural elements ofthe peptide, with the thiolactone C-terminus inhibiting virulence inother sub-groups. The un-modified N-terminus acts as the signal toup-regulate virulence gene expression in the sub-group that synthesisedit, but only in conjunction with the C-terminus, which is also required.The presence of a truncated peptide comprising the C-terminal 5 aminoacids with thiolactone linkage suppresses not only the other threesub-groups, but also the strain that produced it. Thus it follows thatan antibody that recognises the N-terminus of the signal peptide, andeffectively displays the C-terminus by leaving it exposed, willeffectively suppress virulence in all S. aureus strains. Antibodies ofthe present invention may therefore be raised against an epitopepresented by the thiolactone molecule as described above or a structuralelement thereof, for example the peptide sequence or the thiolactonemoiety.

In certain preferred embodiments of the invention, the antibodies arescAbs, in particular scAbs that are obtained from E. coli clonesdesignated as XL1-Blue G3H5, G3B12, G3G2 and/or G3H3. The clones havebeen deposited at NCIMB, Aberdeen, UK on 18 Mar. 2003 under the terms ofThe Budapest Treaty under the following accession numbers: G3H5deposited as NCIMB-41167, G3B12 deposited as NCIMB-41168, G3G2 depositedas NCIMB-41169 and G3H3 deposited as NCIMB-41170. The strains may becultivated in an appropriate growth media such as LB media supplementedwith 100 μg/ml ampicillin, optionally supplemented with 12.5 μg/mltetracycline, and/or 1% glucose, under standard conditions of 37° C. inair.

Bacterial signalling molecules are being discovered in every organismfor which they are searched. It seems to be a ubiquitous system,applicable to every species. The main differences are that all gramnegative (gram −ve) bacteria use homoserine lactone-based molecules, andgram positive (gram +ve) bacteria use (modified) small peptides. Manygram negative and gram positive organisms such as Vibrio harveyi andBascillus anthracis (Jones, M. B. and Blaser, M. J.) also use a smallboron-containing organic molecule AI-2 (AutoInducer-2) which, likehomoserine lactones, is derived from S-Adenosylmethionine. Previous workin this field has concentrated on mimicking signal molecules with onesthat are recognised but that do not function, i.e. no pathogenicswitching, or on blocking the various receptor systems. Thedisadvantages of these methods are principally that resistance can bedeveloped to the mimic or block and the ‘real’ signal molecule is stillthere and will compete for binding. In addition, some bacterialsignalling molecules e.g. homoserine lactones are virulence factors intheir own right, and can directly cause immuno-suppression of the host(i.e. patient). The essence of the present invention is to target theactual signal molecule, and this can be applied to all bacterialcell-to-cell signalling systems (gram negative and gram positive). Thisapproach has a key and important advantage over all previous efforts inthe field in that the bacteria will not recognise that they are beingattacked, they will simply detect that that they are alone. There willnot be any selective pressure for resistance.

According to a second aspect of the present invention, there is provideda pharmaceutical composition comprising an antibody as defined in thefirst aspect of the invention.

Such compositions may be prepared by any method known in the art ofpharmacy, for example by admixing the active ingredient with acarrier(s), diluent (s) or excipient(s) under sterile conditions.

The pharmaceutical composition may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route. Such compositions maybe prepared by any method known in the art of pharmacy, for example byadmixing the active ingredient with the carriers) or excipient(s) understerile conditions.

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; as powders orgranules; as solutions, syrups or suspensions (in aqueous or non-aqueousliquids; or as edible foams or whips; or as emulsions)

Suitable excipients for tablets or hard gelatine capsules includelactose, maize starch or derivatives thereof, stearic acid or saltsthereof.

Suitable excipients for use with soft gelatine capsules include forexample vegetable oils, waxes, fats, semi-solid, or liquid polyols etc.

For the preparation of solutions and syrups, excipients which may beused include for example water, polyols and sugars. For the preparationof suspensions oils (e.g. vegetable oils) may be used to provideoil-in-water or water in oil suspensions.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research, 3 (6),page 318 (1986).

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils. For infections of theeye or other external tissues, for example mouth and skin, thecompositions are preferably applied as a topical ointment or cream. Whenformulated in an ointment, the active ingredient may be employed witheither a paraffinic or a water-miscible ointment base. Alternatively,the active ingredient may be formulated in a cream with an oil-in-watercream base or a water-in-oil base. Pharmaceutical compositions adaptedfor topical administration to the eye include eye drops wherein theactive ingredient is dissolved or suspended in a suitable carrier,especially an aqueous solvent. Pharmaceutical compositions adapted fortopical administration in the mouth include lozenges, pastilles andmouth washes.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or enemas.

Pharmaceutical compositions adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable compositions wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalationinclude fine particle dusts or mists which may be generated by means ofvarious types of metered dose pressurised aerosols, nebulizers orinsufflators.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or sprayformulations.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solution which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation substantially isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Excipients which may beused for injectable solutions include water, alcohols, polyols,glycerine and vegetable oils, for example. The compositions may bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carried, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets.

The pharmaceutical compositions may contain preserving agents,solubilising agents, stabilising agents, wetting agents, emulsifiers,sweeteners, colourants, odourants, salts (substances of the presentinvention may themselves be provided in the form of a pharmaceuticallyacceptable salt), buffers, coating agents or antioxidants. They may alsocontain therapeutically active agents in addition to the substance ofthe present invention.

Dosages of the pharmaceutical compositions of the present invention canvary between wide limits, depending upon the disease or disorder to betreated, the age and condition of the individual to be treated, etc. anda physician will ultimately determine appropriate dosages to be used.

Such compositions may be formulated for human or for veterinarymedicine. The present application should be interpreted as applyingequally to humans as well as to animals, unless the context clearlyimplies otherwise.

According to a third aspect of the invention, there is provided a methodfor the treatment of bacterial infection of a subject, the methodcomprising administration of an antibody of the first aspect of theinvention to the subject.

Examples of bacteria found to cause disease states are shown in Table 1.Methods of this aspect of the invention therefore extend to a method oftreatment of an infection by a strain of bacteria as shown in Table 1 ina subject. In a preferred embodiment of the invention, there is provideda method of treatment of an infection of Pseudomonas aeruginosa in asubject.

Therapeutic substances of the present invention may be used in thetreatment of a human or non-human animal. The treatment may beprophylactic or may be in respect of an existing condition.

The antibody will usually be supplied as part of a sterile,pharmaceutical composition which will normally include apharmaceutically acceptable carrier. This pharmaceutical composition maybe in any suitable form, (depending upon the desired method ofadministering it to a patient).

It may be provided in unit dosage form, will generally be provided in asealed container and may be provided as part of a kit. Such a kit ofparts would normally (although not necessarily) include instructions foruse. It may include a plurality of said unit dosage forms.

The methods of the invention can be applied to short or long-term, acuteor chronic illness/disease, and is effective against most or allbacterial pathogens of plants, animals, including humans. The inventioncan also be used as a prophylactic treatment for the prevention ofdisease onset in individuals at risk of or from exposure to pathogenicbacteria. The invention also has the potential to limit or prevent thedown-regulation of the immune system that results from many infections,and is of particular concern with patients suffering from cancer, cysticfibrosis, AIDS and other immuno-suppressive conditions. Furthermore, asthe methods of the invention are directed particularly at bacterial cellsignalling molecules, and not primarily at the bacterial cellsthemselves, there will be no selective pressure exerted on bacterialpopulations to develop resistance to the treatments described.

The antibody may be administered to infected patients in order tomodulate and reduce bacterial infection. This can include inhalation ofthe antibody in an aerosol by cystic fibrosis patients to increase lifeexpectancy.

In yet another embodiment the antibody is administered toimmuno-suppressed patients in order to increase immuno-competence.

In yet another embodiment conjugates of cell signalling molecules toimmunogenic proteins can be administered to individuals or patients inorder to stimulate an immune response against the signalling moleculeresulting in the generation of neutralising antibodies.

In yet another embodiment the antibody is used as an immuno-diagnosticreagent to detect the presence of, and/or pathogenic status of potentialpathogens, for example in the bloodstream or pleural fluids of patients.

In yet another embodiment the antibody is used as an immuno-capturereagent to selectively remove bacterial cell signalling molecules frompatient's blood in a form of dialysis.

In yet another embodiment alternative methods can be applied to theremoval of bacterial cell-cell signalling molecules from the blood of apatient with a view to modulating the pathogenicity and virulence ofinfecting micro-organisms. This can be achieved with other naturalreceptors or molecules based on natural receptors that bind to saidsignal molecules. Alternatively non-natural receptors can be appliedsuch as molecularly imprinted polymers (MIPs). This class of receptorhave already been shown to be able to bind specifically to smallmolecular weight bio-molecules such as drugs (Hart et al., 2000) andsteroids (Whitcombe et al., 1995; Ramstrom et al., 1996; Rachkov et al.,2000). In a further alternative dialysis can be achieved by thenon-specific removal of all small molecular weight molecules from thepatient's blood as is kidney dialysis.

In yet another embodiment the receptor may have catalytic or enzymaticactivity, and be able to convert the cell signalling molecule into aform that is no longer recognised by the target organism, or no longerresults in pathogenic switching.

In yet another embodiment the antibody is used in one or more of theabove applications in combination, or in combination with othertherapies, for example antibiotics, to provide additive and enhancedtherapeutic regimes, disease monitoring and treatment management.

The antibodies (or equivalent) of the present invention could beadministered to treat bacterial infection, or used as a preventativemeasure for those at high risk of infection. In the case where infectionalready exists, the antibodies may be administered alone or incombination with anti-bacterial antibodies or antibiotics or otheranti-microbial treatments. Administration of anti-HSL antibodies inconjunction with other therapies may allow the use of shorter courses orlower doses of therapeutics, so decreasing the risk of resistancearising and improving patient compliance.

According to a fourth aspect of the invention there is provided anantibody as defined in the first aspect for use in medicine.

According to a fifth aspect of the invention, there is provided the useof an antibody as defined in the first aspect in the preparation of amedicament for the treatment of bacterial infection.

According to a sixth aspect of the invention, there is provided a methodof screening a population of specific binding molecules for ananti-bacterial specific binding molecule, the method comprisingconjugating a bacterial lactone signal molecule to a carrier moleculeand using the conjugate so formed to identify a specific bindingmolecule that specifically binds to the conjugate from the population ofspecific binding molecules.

Such methods are therefore a means for identifying a specific bindingmolecule that can be used as an anti-bacterial agent, for example in thetreatment of a bacterial infection. The specific binding molecule is anantibody or a fragment thereof, for example a monoclonal antibody, or apolyclonal antibody. Suitably the carrier molecule is a protein asdescribed above. The population of specific binding molecules may be aphage display library.

Specific binding molecules identified by a method of the presentinvention may be used in medicine or a method of treatment as describedabove. The specific binding molecules may further be used in thepreparation of a medicament for the treatment of a bacterial infection.

Such methods therefore extend to uses of a bacterial lactone signalmolecule to screen a population of specific binding molecules in orderto identify a specific binding molecule that specifically binds to saidbacterial lactone signal molecule.

According to a seventh aspect of the invention, there is provided amethod of treatment of a bacterial infection of a subject, the methodcomprising isolation of a bacterial lactone signal molecule in a samplefrom said subject and using said bacterial lactone signal molecule toscreen a population of specific binding molecules for an anti-bacterialspecific binding molecule to identify a specific binding molecule thatspecifically binds to the signal molecule, and administering saidspecific binding molecule so identified to a patient in need thereof.

Such methods permit the identification of specific binding moleculesdirected against the infecting bacterial organisms whose signallingmolecules are found in the sample. The sample may be of blood, saliva,tissue, cerebro-spinal fluid, tears, semen, mine, faeces, pus, skin, ormucous secretions. Samples of blood may be of whole blood, or offractionated blood, for example, blood plasma. Tissue samples may be abiopsy of any infected or potentially infected tissue or organ. Samplesmay also be taken from wounds or sites of injury or infection orpotential infection. Samples of fluid from the lungs or the contents ofthe stomach or the intestines may also be used.

Preferred features for the second and subsequent aspects of theinvention are as for the first aspect mutatis mutandis.

Other objects, features and advantages of the present invention,including but not limited to related applications in plant and animalhosts, will be apparent to those skilled in the art after review of thespecification and claims of the invention.

It will be apparent to those of ordinary skill in the art that thecompositions and methods disclosed herein may have application across awide range of organisms in inhibiting, modulating, treating ordiagnosing disease or conditions resulting from infection. Thecompositions and methods of the present invention are described withreference to Pseudomonas aeruginosa, but it is within the competence ofone of ordinary skill in the art to apply the objects herein to otherspecies.

The invention will now be further described by reference to thenon-limiting example and figures detailed below.

DESCRIPTION OF FIGURES

FIG. 1A shows the chemical structures of the three representativeclasses of homoserine lactone bacterial cell signalling molecules. Thesediffer in the substitution at position C3, and vary within each class bythe length of the acyl side-chain (typically n=0 to n=10). In addition,there may be a cis-bond present within the acyl chain. FIG. 1B shows thestructures of i) pro AI-2, the immediate precursor of AI-2, ii) theboron-containing active AI-2 molecule and iii) the reactive pro-AI-2hapten used to make conjugates; and FIG. 1C shows examples ofthiolactonc peptide signalling molecules used by i) Staphylococcusaureus Group I, ii) S. aureus Group II, iii) S. aureus Group III and iv)S. aureus Group IV.

FIG. 2 illustrates the genetic regulation of quorum or cell densitydependent sensing. The cell signalling mechanism consists of twocomponents: 1) the I gene (lasI and rhlI) homologues synthesiseincreasing quantities of bacterial cell signalling molecules (HSLs)throughout growth (hence quorum sensing), and 2) the concentrationdependant binding of signalling molecules to a cognate R proteinhomologue (encoded by lasR and rhlR) which in turn can switch on aseries of particular genes (operon), allowing bacteria to co-ordinate adensity dependent phenotypic switch (eg. virulence, swarming).

FIG. 3 illustrates the principals of the bioluminescent reporter geneassay using plasmid pSB1075. A reduction in light output is indicativeof the successful blocking of bacterial cell signalling.

FIG. 4 shows a comparison of the abilities of HSL-specific andirrelevant single-chain antibodies immobilised onto an inert columnmatrix to remove HSL from solution by immuno-affinity capture. Columneluates were applied to an E. coli surrogate of Vibrio fischeri(JM107-pSB401) and the effect of residual HSL in the eluates determinedfrom the subsequent stimulation of the bacterial cultures to fluoresceas measured by RLU. ScAbs G3B12 and 0302 are HSL-specific, anti-VZV isspecific for a viral protein, anti-Paraquat and anti-Atrazine arespecific for herbicides with molecular weights similar to HSL molecules,and the Resin control contained no immobilised scAb. Data represents themeans of three replicate samples from two separate assays. Standarderrors are indicated.

FIG. 5 shows the inhibitory effects of specific and irrelevantsingle-chain antibodies on the dDHL-mediated stimulation of an E. colisurrogate of Ps. aeruginosa (JM109-pSB1075) as measured bybioluminescence output. Data is given for the HSL-specific scAb 03115(●), a non-specific control scAb (▾) (specific for a pathogenicbacterial surface protein), and in the absence of scAb (◯). Data pointsrepresent the means of three replicate samples from replicate assays.

FIG. 6 shows the inhibitory effects of specific and irrelevantsingle-chain antibodies on the tDHL-mediated stimulation of an E. coilsurrogate of Ps. aeruginosa (JM109-pSB1075) as measured bybioluminescence output. Data is given for three HSL-specific scAbs; G3H3(●), G3G2 (▪) and G3B12 (□), for the irrelevant anti- V scAb (◯)(specific for a pathogenic bacterial surface protein), and in theabsence of scAb (∇). Data points represent the means of three replicatesamples from replicate assays.

FIG. 7 shows the inhibitory effects of specific and irrelevant(non-specific) single-chain antibodies on the BBL-mediated stimulationof an E. coli surrogate (JM109-pSB406) of Ps. aeruginosa Rrhl system(short-chain HSL responsive) as measured by bioluminescence output after60 min (FIG. 7A) and 150 min (FIG. 7B). Data is presented for G3H5, G3B12 and G3H3 antibodies, a non-specific control antibody (specific for apathogenic bacterial surface protein), and in the absence of antibody(PBS buffer only). Data points represent the means of three replicatesamples from replicate assays.

FIG. 8 shows the slow kill nematode assay demonstrating the ability ofG3H5 and G3B 12 antibodies to protect nematodes against infection by thebacterial pathogen Pseudomonas aeruginosa strain PA14 (FIG. 8A) and Ps.aeruginosa strain PA01 (FIG. 8B).

FIG. 9 shows competition ELISA data for anti-peptide scAb YST-1 bindingto BSA control, or BSA-peptide conjugate in the presence or absence offree peptide ‘YSTGGAGSGG’ or free thiolactone peptide Agr-D1 (see FIG.1C).

FIG. 10 shows Table 1. Table 1 lists various bacterial phenotypes, withthe cell signalling molecules and regulatory elements of the quorumsensing system that regulate them, for a range of organisms.

FIG. 11, comprising panels A-C, shows Tables 2, 3, and 4, respectively.FIG. 11 a depicting Table 2 shows a summary of the sensitivities (IC₅₀)of anti-AHL scAbs to free antigen (dDHL-COOH) and to two AHL analogues(tDHL and OHHL) in competition with dDHL-BSA as determined bycompetitive inhibition ELISA. FIG. 11 b depicting Table 3 shows acomparison of the kinetics of two anti-AHL scAbs binding to immobiliseddDHL-BSA conjugate as determined by Surface Plasmon Resonance using aBIAcore 2000 instrument. The association constants (ka), dissociationconstants (kd) and affinity constants (KA, KD) are given. FIG. 11 cdepicting Table 4 shows a summary of the sensitivities (IC₅₀) ofanti-HSL clones derived from chain-shuffling to various HSLs. Enclosedin brackets ( ) below each new clone is the designation of the clonefrom which it was derived. The degree of increased sensitivity toantigen of new clones over the starting clone is given in brackets ( )where applicable. Data compare the binding to fee HSLs in competitionwith dDHL-TG conjugate as determined by competition ELISA.

FIG. 12 shows Table 5. Table 5 shows the effects of anti-HSL scAbs inreducing the expression of the virulence factor elastase by Ps.aeruginosa. Data represent the ratio of clearance zone to colony area,expressed as a percentage compared to the PBS control (100%).

EXAMPLE 1

The examples described herein relate to Vibrio fisheri and Pseudomonasaeruginosa. These are given only as an example, the scope of theinvention not being limited to the example but including all bacterialcell-to-cell signalling molecules that directly or indirectly regulateexpression of genes involved in virulence or pathogenicity, and alsoincluding other signal molecule-induced phenotypic changes to bacterialcells such as but not limited to bioluminescence.

A derivative of a HSL was synthesised (designated dDHL-COOH), having atwelve-carbon acyl chain acting as a ‘linker’, and terminating in acarboxylic acid group (see FIG. 1). This was conjugated, via thecarboxylic acid group, to the carrier proteins Bovine Serum Albumin(BSA) and Keyhole Limpet Haemocyanin (KLH) to produce dDHL-BSA anddDHL-KLH. Briefly, 50 mg BSA or KLH was dissolved in 1.67 ml water, andto it added 3.3 ml of 2 mM KH₂PO₄ at pH 8.5, all at 4° C. To this, 1.05ml dry dimethylformamide (DMF) was added drop-wise while stirring. Tenmilligrams of activated N-hydroxysuccinimide ester of dDHL-COOH wasdissolved in 100 μl dry DMF, and again added slowly to the carrierprotein solution at 4° C. The reaction mixture was stirred well andallowed to stand for 24 h at 4° C. The conjugated material was thendialysed against 4×1 liter water, and conjugation confirmed by MALDITOFmass spectroscopy.

The term ‘linker’ refers to any chemical group used to allow attachmentof the hapten (antigen) to a (preferably) immunogenic carrier moleculesuch that the hapten is displayed away from the surface of the carrier.

In alternative objects of the invention, other carrier molecules such asmagnetic beads or biotin, and other linkers and conjugation strategiescan be employed. The two conjugated forms of dDHL were then used toscreen an antibody phage display library. Briefly, the library wasscreened for a total of 3 rounds of bio-panning. In each round adDHL-conjugate was immobilised onto a solid support and incubated withthe library of phage-antibodies for sufficient time for phage-antibodiesrecognising the conjugate to bind. Unbound phage were removed bystringent washing with PBS (Phosphate Buffered Saline) and PBS-Tween,and the remaining bound phage eluted by incubation at low pH (round 1).Eluted phage were then infected into E. coli bacteria and amplified bymethods familiar to those practised in the art. The resulting amplifiedlibrary of enriched clones was then used for the following round ofpanning. In order to reduce the numbers of clones selected thatrecognised the carrier protein, the immobilised conjugate (dDHL-BSA ordDHL-KLH) was alternated with successive rounds of selection. In orderto bias selection in favour of clones recognising a specific HSL, thechosen HSL (dDHL-COOH) was used to competitively elute phage-antibodiesduring rounds 2 and 3, rather than low pH. Individual phage clones fromround 3 were screened by ELISA: Each clone was assayed initially for theability to bind to each of the dDHL-conjugates and to the carrierproteins alone. Those clones able to bind both conjugates but unable tobind either carrier protein were further assayed to identify those whosebinding to conjugate could be inhibited by the presence of freedDHL-COOH in solution. The antibody variable region genes from thosephage clones found to bind to free dDHL-COOH were sub-cloned into asoluble expression vector (pIMS 147), and produced as solublesingle-chain antibody fragments (scAb) comprising the variable heavy andlight chain domains joined by a flexible peptide linker, and a kappaconstant domain from a human antibody. Quantification of the binding ofsoluble scAb to free HSLs was determined by competitive inhibition ELISASamples containing a constant concentration of each selected scAb (withrespect to 1 microgram per ml dDHL-BSA) were incubated with a range ofconcentrations of free dDHL-COOH (or dDHL-conjugate) for 1 h, thenapplied to an ELISA plate coated with dDHL-BSA. After 1 h incubation,unbound scAb was washed off and any scAb remaining bound to theimmobilised conjugate detected with enzyme-labelled anti-human kappaantibody. The sensitivity of scAb for free dDHL-COOH, and crossreactivity with other HSLs (tDHL and OHHL) was determined from theconcentration of free antigen that reduced the binding of scAb (withoutfree antigen) to dDHL-BSA by 50% (IC₅₀) (Table 2).

The binding kinetics for anti-HSL scabs binding to dDHL-BSA wasdetermined using a BIAcore 2000 (BIAcore, Sweden). A CM5 chip wasactivated with 0.2 M EDC[1-3-(3-dimethyl-aminopropyl)carbodiimide-HCl]/0.05 M NHS(N-hydroxy-succinimide), and dDHL-BSA or BSA alone coupled to the chipin 10 nM Na-acetate at pH 3.5 or 4.5 respectively. A series of 10concentrations of scAb (100 to 1000 nM) were assayed in duplicate in HBSbuffer at a flow rate of 20 microliters/min. Between samples the chipwas regenerated with 20 microliters 100 mM NaOH. Kinetics weredetermined using the BIAevaluation 3 software package (Table 3).

The ability of the scAb G3B12 to bind to OHHL was further assessed byimmobilising scAb to nickel-sepharose beads in a column via a6×histidine tag, and passing a solution of OHHL through the column. AnyOHHL bound by the scAb and retained on the column was subsequentlyeluted. The concentration of OHHL in the column flow though (i.e.unbound) and that bound and later eluted were determined.

The ability of the scAbs to bind to HSLs and to modulate the response ofbacteria to AHLs was determined using E. coli strains JM107 containingthe plasmid pSB401 (Vibrio fischeri response surrogate) and JM109containing the plasmids pSB406 and pSB406 (Pseudomonas aeruginosaresponse surrogate). The reporter plasmids contain the HSL responseregulator genes luxR (pSB401), lasR (pSB1075, responsive to long-chainHSLs) or rhlR (pSB406, responsive to short-chain HSLs), and the luxIpromoter region, which together with exogenous HSLs activates expressionof the luxCDABE gene fusion (the luminescence structural genes) fromPhotorhabdus luminescens. Under the appropriate growth conditions thesecells are induced to emit light in response to the presence ofextra-cellular HSLs, the intensity of light emitted being proportionalto the concentration of HSL.

Soluble scAbs from clones selected from the library were expressed usingpublished protocols (Strachan et al., 1998). During immobilised metalaffinity chromatography purification (IMAC), scAb was not elated fromthe nickel-sepharose column. A series of additional scAbs withspecificities to irrelevant antigens were also expressed and immobilisedonto nickel-scpharose columns to act as controls. Five hundredmicroliters of 10 nM OHHL was applied to each column and incubated for 1hour at 4° C. Columns were centrifuged at 40 g for 15 s and the flowthrough collected. Any bound OHHL was eluted with 250 microliters 1 MNaCl. The original flow through was re-applied and incubated as before,the flow through collected and bound HSL eluted with 1 M NaCl.

Samples of HSL solution prior to and after passage through theimmobilised scAb column were applied to E. coli JM107 pSB401 culturesand the light emitted measured with a luminometer. Appropriate controlexperiments were carried out using a column to which no scAb had beenimmobilised, and three additional columns including scAb withspecificity's for irrelevant antigens. Cells were grown shaking at 37°C. for 18 h in-LB medium containing tetracycline. One milliliter of theculture was inoculated into 100 ml LB tetracycline medium and grown at37° C. until an OD 600 nm 0.2 was achieved. One hundred microliters ofthe culture was applied to replicate wells of a 96-well black bio-assayplate, and an equal volume of HSL solution added. HSL solutions were 10nM OHHL (positive control), milli-Q water passed through anickel-sepharose column (resin control), or the flow through frompassing 10 nM OHHL over columns containing immobilised scAb as describedabove. Plates were incubated at 37° C. for 2 h with shaking, andluminescence read using an Anthos LUCY1 luminometer for 1 s (FIG. 4).

The ability of the scAbs to reduce bacterial responses to long-chainHSLs was assessed with an HSL-inducible luminescence reporter bioassayover a period of 3.0 h using E. coli strain JM109-pSB1075. This strainis essentially as described for JM107-pSB401, the difference being thatplasmid pSB1075 includes the lasR of Pseudomonas aeruginosa in place ofthe luxR of Vibrio fischeri. Single colonies of JM109-pSB1075 wereinoculated into 10 ml LB broth with antibiotic and incubated overnightat 37° C. Two hundred microliters of overnight culture were inoculatedinto 10 ml fresh medium and incubated at 37° C. with shaking to OD 600nm 0.2. HSL was added to the cultures (dDHL-COOH at 20 nM final conc'nor tDHL at 50 nM final conc'n) and one hundred microliters of culturewas added to triplicate wells of a black 96 well plate. LB medium wasadded to negative controls. Either 50 microliters PBS or 50 microlitersscAb at 2 mg/ml was added to each well and the plate incubated furtherfor three hours shaking at 37° C., after which time luminescence wasmeasured at 30 min intervals and the effect of scAb on cell signallingdetermined (FIGS. 5 and 6). The data demonstrates the ability ofanti-HSL antibodies to cross react with structurally differenthomoserine lactone signal molecules, and to reduce or eliminate theresponse of a Ps. aeruginosa surrogate to extra-cellular HSL.

The ability of the scAbs to reduce bacterial responses to short-chainHSLs was assessed in a similar way to that described above. Thebioluminescence reporter system used E. coli strain JM109 with thereporter plasmid pSB406, including the rhlR response element regulator.The signal molecule BHL (as acyl-HSL in FIG. 1 but 4 carbon side chain)was added to the E. coli cultures to 50 nM final concentration (anequivalent volume of LB medium was added to negative control cultures),and 100 μculture added to triplicate wells of the assay plate. Either 50μl scAb at 100 nM or 50 μl PBS was then added, and plates incubated asdescribed earlier. Measurements of luminescence were taken after 60 minand 150 min (FIG. 7).

EXAMPLE 2

To assess the ability of anti-HSL scabs to afford protection to animalsagainst pathogenic Ps. aeruginosa, a ‘slow-killing’ assay using thenematode C. elegans was employed. This assay is based on the killing ofthe worms following establishment of a Ps. aeruginosa infection in theanimal's gut.

Ps. aeruginosa strain PA14 was infected into 5 ml LB broth on day 1 andincubated overnight at 37° C. On day 2, 1% of the overnight culture wasinoculated into 5 ml fresh LB with 100 μl scAb at 100 nM broth andincubated at 37° C. to OD 600 nm of 0.4. Ten microliters bacterialculture was spotted onto the centre of NG enriched peptone agar plates(nematode growth media), together with 50 μl scAb at 120 nM and platesincubated overnight at 37° C. On day 3 (pm) an additional 50 μl scAb(120 nM) was spotted onto the plates and incubation continued overnight.On day 4 (am) the plates were transferred to room temperature (˜20° C.)and a further 50 μl scAb added. On day 5 (am), 50 μl scAb was added asbefore, and 20-5-adult worms added directly onto the bacterial lawn(time=0 h). Supplementary additions of scAb were made at time=26, 50 and76 h. The numbers of dead worms were determined at intervals over thefollowing 3 days (FIG. 8 a). Worms were considered dead when non-motileand not responsive to touch by a fine wire pick.

For control plates either PBS or an irrelevant scAb (specific for anunrelated target antigen) were used, or worms were grown on E. colistrain OP50.

A second assay was also carried out using Ps. aeruginosa strain PA01(Darby et al., 1999). This strain is used primarily for ‘paralytickilling’ (toxin production), but it also suitable, though less effectivethan PA14, for slow killing infection studies as in this example.

The assay was carried out as described above with the followingmodifications. Additions of scAb throughout were at 100 nMconcentration. On day 3, 50 μl scAb was spotted onto plates in themorning and afternoon. After the second addition, the plates weretransferred to room temperature and incubated overnight. Worms and scAbwere applied on day 4 (time =0 h), and only two supplementary scAbadditions made at t=30 and 60 h (FIG. 8 b).

EXAMPLE 3

Ps. aeruginosa produces several extracellular products that, aftercolonisation, can cause extensive tissue damage. One of these, elastase,is essential for maximum virulence of Ps. aeruginosa during acuteinfection. The production of elastase is under the control of the lasI/Rquorum-sensing cascade. The detection of elastase production cantherefore be used as an indicator of the capacity for virulence of thebacterial population.

Four microliters Ps. aeruginosa strain PA14 at 10 sup 5 CFU per ml (lowOD) or 10 sup 8 CFU per ml (high OD) were inoculated onto agar platescontaining 1% elastin, 0.5% lab lemco powder, 1% peptone, 0.5% sodiumchloride and 1.5% agar, and incubated at 37° C. for 5 days. Growth tolow OD discourages pathogenic switching, whereas growth to high ODencourages pathogenic switching. Fifty microliters of scAb (200 nM) wasadded to the plates together with the bacteria, and additionalapplications of the same volume made at 24 h intervals throughout theassay.

The diameter of the bacterial colonies and the surrounding clear zones(indicative of lysis of elastin by elastase) were measured daily, andthe elastolytic activity of the colonies determined as a ratio of theclear zone area to bacterial colony area. Again, 3-4 replicates pertrial were performed and E. coli XL1-Blue was be used as a negativecontrol (Table 5).

EXAMPLE 4

In order to isolate anti-HSL antibodies with higher affinity forantigen, and to direct specificity towards particular HSL variants,affinity maturation was performed on clone G3B12. Phagemid DNA wasisolated from the G3B12 bacterial clone, and the variable light chaingene amplified by PCR using a 5′ oligonucleotide primer, LINKER-REVcomprising the last 30 bases of the 45 base-pair flexible linker region(5′-GGCGGAGGTGGCTCTGGCCGGTAGTGC-3′) and a 3′ primer gIII-FOR(5′-GAATTTTCTGTATGAGG-3′), specific for the phage minor coat proteingene gIII. Product of the correct size (˜380 bp) was electrophoresed ina 1% agarose gel, excised, and purified. In a similar way, phagemid DNAcontaining the entire human naïve library from which the original clonewas isolated was prepared. The whole repertoire of variable heavy chaingenes was amplified using the 5′ primer AHl-REV(5′-AAATACCTATTGCCTACGGC-3′) specific for the pelB leader sequence, andthe 3′ primer LINKER-FOR encoding the first 30 bases of the linkerregion (5′-AGAGCCACCTCCGCCTGAACCGCCTCCACC-3′). Product of the correctsize (˜400 bp) was purified as above. The repertoire of VH genes wasthen combined with the monoclonal VL gene by linking PCR using thecomplementary 15 bases of the centre of the linker region that wascommon to both primary PCR products. The new library was amplified byaddition of the primers AH1-REV and gIII-FOR to the linking PCR reactionafter 4 cycles, and a further 25 cycles performed. The amplified DNA wasdigested with the restriction enzymes NcoI and NotI, and ligated into asimilarly digested and purified phagemid vector. Ligated and re-purifiedDNA was finally transformed into E. coli strain TG1 cells byelectroporation and plated in the conventional manner.

Phage antibodies were rescued with helper phage as before, and appliedto immunotubes coated with dDHL-BSA conjugate, and allowed to bind.Unbound phage were poured off, and weak/non-specific binders removed bymultiple wash steps with PBST and PBS. Conjugate specific phage werethen eluted with low pH triethylamine and neutralised. These wereinfected into fresh log-phage TG1 cells, rescued, and amplified againfor following rounds of selection (pans). For the successive pans, theimmobilised conjugate was alternated between dDHL-BSA and dDHL-TG.During the third round of panning, the bound phage from the shuffledlibrary were eluted from duplicate pans with either free dDHL or withBHL (butyrylhomoserine lactone).

After 3 rounds of panning, monoclonal phage-antibodies were screened fordesired binding characteristics. Individual phage clones from round 3were screened by ELISA: Each clone was assayed initially for the abilityto bind to each of the dDHL-BSA and KLH conjugates and to the carrierproteins alone. Those clones able to bind both conjugates but unable tobind either carrier protein were further assayed to identify those whosebinding to conjugate could be inhibited by the presence of free dDHL orHSL in solution. The antibody variable region genes from those phageclones found to bind to free BHL/dDHL were sub-cloned into a solubleexpression vector (pIMS 147), and produced as soluble single-chainantibody fragments (scAb) comprising the variable heavy and light chaindomains joined by a flexible peptide linker, and a kappa constant domainfrom a human antibody. Quantification of the binding of soluble scAb tofree HSLs was determined by competitive inhibition ELISA. Samplescontaining a constant concentration of each selected scAb (with respectto 1 microgram per ml dDHL-TG) were incubated with a range ofconcentrations of free HSL for 1 h, then applied to an ELISA platecoated with tDHL-TG. After 1 h incubation, unbound scAb was washed offand any scAb remaining bound to the immobilised conjugate detected withenzyme-labelled anti-human kappa antibody. The sensitivity of scAb forfree HSL, and cross reactivity with other HSLs was determined from theconcentration of free antigen that reduced the binding of scAb (withoutfree antigen) to dDHL-TG by 50% (IC₅₀) (Table 4).

EXAMPLE 5

Two conventional peptides, YST-1 (YSTGGAGSGG) and YST-2 YSTASGGASS weresynthesised, together with a third version, YST-3, with biotinylation^(^ denotes site of biotinylation) of the penultimate C-terminal lysineside chain (YSTAGGSGAK^S). A fourth thiolactone peptide YSTC*DFIM*(Agr-D1) was also synthesised, where * denotes residues connected by thethiolactone ring (see FIG. 1 c). Some of YST-1 was conjugated to BSA andYST-2 to bovine thyroglobulin (TG) via the C-terminus using1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) by conventionalconjugation chemistry.

A human naïve antibody library was panned for four rounds againstalternating immobilised YST-1 and YST-2 conjugates essentially asdescribed previously. After binding phage-antibodies for 1 h, unboundphage were poured away and weakly bound phage removed by extensivewashing with PBST followed by PBS. Bound phage were eluted withthethylamine and neutralised, then infected into log phase E. coli TG1cells. The enriched phage were amplified by rescuing with helper phage,then purified and concentrated by polyethylene glycol precipitationready for the next round of selection. Following washing of phage boundto YST-1/2-conjugates in rounds 3 and 4, phage were eluted with asolution of YST-3. Those phage binding to the biotinylated peptide werecaptured by streptavidin coated paramagnetic beads and immobilised witha magnet. After further wash steps, the bound phage were added directlyto TG1 Cells and allowed to infect as before.

Following four rounds of selection, monoclonal phage-antibodies werescreened by ELISA for binding to YST-1 and YST-2 conjugates, and to BSAand TG carrier proteins alone. The scFv genes of those clones that boundonly to both conjugates were sub-cloned into the pIMS-147 scAb solubleexpression vector and transformed into E. coli XL1-Blue cells. Thesecells were expressed, and soluble scAb extracted from the bacterialperiplasmic space and purified by Nickel affinity chromatography.Purified scAb was then further assayed for binding to free(non-conjugated) peptide and to the thiolactone-peptide autoinducerAgr-D1, in competition with immobilised peptide conjugates by ELISA.Signal reduction in the presence of peptide compared to binding toconjugate alone indicates that scAb is recognising the YST-epitopecommon to all peptides (FIG. 9).

EXAMPLE 6

In order to generate antibodies against the AI-2 target, both the freeAI-2 molecule and a conjugated form are required. It is not (considered)possible to isolate AI-2 in a pure form. In nature, AI-2 is formed bythe (spontaneous) reaction of pro-AI-2 (FIG. 1( b)) with boric acid. Invitro, pro-AI-2 will also react with boric acid to yield active AI-2,however this is not suitable for conjugation and antibody selection. Aderivative of pro-AI-2 can be synthesised whereby the methyl group isreplaced by a linker e.g. an acyl chain, with a terminal reactive groupe.g. carboxylic acid. Any structure that includes a terminal reactivegroup suitable for chemical conjugation or cross-linking to a carrier,and that will result in the core pro-AI-2 moiety being displayed clearof the carrier surface, can be used as a linker. The reactive pro-AI-2is conjugated to preferably two different carriers as described in thesection ‘summary of the invention’.

A library of potential receptors (e.g. an antibody library displayed onphage) is applied to an immobilised conjugate in the presence of boricacid (preferably >10 μM, pH 6.0-8.0) in order to yield immobilised AI-2conjugate. Phage-antibodies (‘phage’) are allowed to bind, and those notrecognising conjugate are removed by washing. Bound phage can be elutedwith high or low pH e.g. triethylamine, or by competitive binding withfree AI-2 or by competitive binding with e.g. biotinylated AI-2 followedby removal with magnetic streptavidin beads. During all stages ofbio-panning except extreme pH elution, borate should be present toensure that the correct structure of AI-2 is maintained. Eluted phageshould be re-infected into host bacteria (E. coil), amplified by growingcells under phage-particle producing conditions and purified for thenext round. Subsequent rounds of selection are carried out as describedfor round one except that, preferably, the immobilised conjugate isalternated.

When sufficient rounds selection have been completed, individual(monoclonal) clones can be assayed for binders to AI-2. It is probablethat at least three rounds of selection will be needed, although AI-2binding clones may be isolated after only one round, or it may benecessary to perform more than three rounds. Polyclonal phage-ELISA canbe performed after each round by methods well known to those familiarwith the art to determine how many rounds are required. Monoclonalphage-antibodies should be produced as described in earlier examples,and assayed for binding to each of the conjugates used for selection andto the respective un-conjugated carriers. Third or fourth conjugate(s)may be used additionally if available. Putative positive clones will beidentified as those binding to all available conjugates in the presenceof borate, but not to carrier molecules alone and preferably not toconjugate in the absence of borate.

As it is possible that the reaction of pro-AI-2 with borate may yieldmore than one species, it will be preferable to demonstrate thatantibodies recognise in particular the correct AI-2 structure. Thiscould be determined by assaying for binding to conjugate in the presenceof free pro-AI-2 and borate. A reduction in binding with increasingconcentrations of free pro-AI-2 is indicative of competitive inhibition.Such antibodies would therefore be expected to be able to modulate theresponse of AI-2 responsive bacteria by binding to extracellular AI-2and rendering it unavailable to cells.

A suitable in vivo model can be found in bioluminescent Vibrio harveyi,a bacterium that bioluminesces in response to AI-2. Strains of V.harveyi that are LuxS⁻ are unable to synthesise DPD (a precursor ofpro-AI-2), and so cannot produce AI-2. They are however able to respondby (increased light output) in response to exogenously added AI-2,either in the form of pro-AI-2 together with borate, or asborate-containing cell-free culture media obtained from LuxS⁺ V. harvei.The addition of borate alone to LuxS⁻ cells or to LuxP⁻ cells (lackingthe natural AI-2 receptor) does not result in any light emission.Potential anti-AI-2 antibodies (‘receptors’) could therefore beidentified as those fulfilling the binding criteria outlined earlier,and also being able to either deplete AI-2 from w.t. V. harvei culturemedia as determined by reduced light emission when added to LuxS⁻ cells,or to quench/prevent/reduce light emission when added to LuxS⁺ cells.

References Cited

U.S. Patent Documents

6,309,651 October 2001 Frank et al.Other Patent Documents

WO 01/26650 April 2001 University of Nottingham WO 01/74801 October 2001University of Nottingham WO 92/01047 October 2001 Bonnert et al.Other References

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1. A method of screening a naive human phage display library for ananti-bacterial monoclonal antibody, comprising: conjugating a bacteriallactone or lactone-derived signal molecule to a first carrier moleculeto generate an enriched library; and screening said enriched libraryagainst the bacterial lactone or lactone-derived signal moleculeconjugated to a second, different, carrier molecule to identify amonoclonal antibody that specifically binds to the free soluble form ofthe bacterial lactone or lactone-derived signal molecule from theenriched library in the presence of conjugated derivatives thereof.
 2. Amethod as claimed in claim 1 in which the lactone signal molecule is ahomoserine molecule or a peptide thiolactone molecule.
 3. A method asclaimed in claim 2 in which the homoserine lactone molecule has ageneral formula selected from the group consisting of:

where n =0 to
 12. 4. A method as claimed in claim 3 in which thehomoserine lactone molecule of general formula I isN-butanoly-L-homoserine lactone (BHL) where n =0,N-dodecanoyl-L-homoserine lactone (dDHL) where n =8, orn-tetradecanoyl-L-homoserine lactone (tDHL) where n =10.
 5. A method asclaimed in claim 3 in which the homoserine lactone molecule of generalformula II is N-(-3-oxohexanoyl)-L-homoserine lactone (OHHL) where n =2or N-(-3-oxododecanoyl)-L-homoserine lactone (OdDHL) where n =8.
 6. Amethod as claimed in claim 3 in which the homoserine lactone molecule ofgeneral formula III is N-(-3-hydroxybutanoyl)-L-homoserine lactone(HBHL) where n =0.
 7. A method as claimed in claim 2 in which thepeptide thiolactone has a general formula (IV) as follows:

where X is any amino acid and n =1 to
 10. 8. A method as claimed inclaim 7 in which the peptide thiolactone molecule is:


9. A method as claimed in claim 1 in which the lactone-derived signalmolecule is a furanosyl borate diester.
 10. A method as claimed in claim9 in which the furanosyl borate diester is Auto Inducer-2 (AI-2),


11. A method as claimed in claim 1 in which the lactone-derived signalmolecule is Pro-AI-2 or a C₁-C₁₀ saturated or unsaturated carboxylicacid derivative thereof


12. A method as claimed in claim 1 in which the antibody is a singlechain antibody (scAb).
 13. A method as claimed in claim 1 in which theantibody is an antibody fragment.
 14. A method as claimed in claim 13 inwhich the antibody fragment is a single chain variable fragment (scFv)or a single domain fragment.